Analysis of amino acids in body fluid by liquid chromotography-mass spectrometry

ABSTRACT

This disclosure provides methods for quantifying individual amino acids in various bodily fluids obtained from a human patient. Also provided are reference ranges for normal amino acid levels in the various bodily fluids (e.g., blood plasma, urine, cerebrospinal fluid, and saliva) and for various age groups (e.g., neonates, infants, children, and adults).

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/241,291 (issued as U.S. Pat. No. 10,962,550), which is a continuationof U.S. application Ser. No. 15/875,850, filed Jan. 19, 2018 (issued asU.S. Pat. No. 10,203,337), which is a continuation of U.S. applicationSer. No. 12/646,666, filed Dec. 23, 2009 (issued as U.S. Pat. No.8,969,089), which is a continuation-in-part of International PatentAppl. No. PCT/US2008/068653, filed Jun. 27, 2008, which claims priorityto U.S. Appl. No. 60/947,338, filed Jun. 29, 2007, each of which ishereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to the detection and analysis ofamino acids, particularly amino acids contained in biological fluids.

BACKGROUND

The identity and amount of amino acids in a patient's body fluid (e.g.,plasma) is important in a patient's health for a number of reasons.Aberrant amino acid levels can be used to diagnose disease or illness.For example, low plasma amino acid levels may occur in patients withcancer, anorexia, arthritis, folliculitis, alcohol abuse, glucagonoma,and/or pregnancy. Patients undergoing stress or depression may also havelow plasma amino acid levels. In particular, depressed patients may bedeficient in phenylalanine, tyrosine, methionine, glycine, tryptophan,and/or taurine. Psychotic patients may have low levels of amino acidssuch as glycine, tryptophan, and/or histidine and elevated levels ofamino acids such as phenylalanine, tyrosine, and/or serine.

Patients with infectious disease and/or fever also may have reducedamino acid levels, although some amino acids in such patients such asphenylalanine may be present at elevated levels. Patients with kidneyfailure may have low levels of amino acids such as tyrosine, threonine,leucine, isoleucine, valine, lysine, and/or histidine. Patients withCrohn's disease, ulcerative colitis, chronic fatigue syndrome may haveabnormally low levels of cystine and glutamine in their plasma.

In addition, amino acid levels that are higher than normal may beindicative of a disease state. For example, elevated plasma amino acidlevels may be observed in patients with liver disease, pancreatitis,heavy metal poisoning, vitamin C deficiency, and/or vitamin Ddeficiency. In particular, patients with Wilson's disease may exhibitelevated levels of tryptophan and histidine. Patients with Cushing'sdisease or gout may exhibit elevated alanine levels. Diabetic patientsmay exhibit elevated levels of valine, leucine, and/or isoleucine.Hyperactive children may exhibit elevated levels of tyrosine andphenylalanine. Patients with Maple Syrup Urine Disease may have elevatedlevels of leucine, isoleucine, and valine in their plasma. As such,methods for analyzing amino acids in body fluids such as plasma areuseful in medicinal and scientific settings.

Traditionally, analytical methods for amino acids have included aderivatization step. During derivatization, the amino acid is reactedwith a derivatizing reagent that facilitates analysis of amino acids inthe sample. Derivatizing agents typically react with the free aminogroups of amino acids in the sample. Common reagents for derivatizingamino acids include isothiocyanates (e.g., phenyl isothiocynate (PITC)),o-phthaldialdehyde (OPA), 2,4-dinitrofluorobenzene (DNFB), andNα-(2,4-dinitro-5-fluorophenyl)-L-alainamide (FDAA). Derivatizing agentsare useful because they may include substituents that facilitateanalysis of the derivatized amino acid. For example, derivatizing agentsmay include chromophores for UV-absorption detection or fluorophores forfluorescent detection.

Derivatized amino acids may be separated and detected by performingchromatography such as liquid chromatography (LC) or gas chromatography(GC), coupled with mass spectrometry (i.e., LC-MS or GC-MS). Aminoacids, however, have diverse chemical structures (e.g., basic, acidic,aromatic, polar, non-polar, etc.), and because of significantdifferences in the chemical structures of various amino acids that maybe present in body fluids, these compounds present a difficult task foranalysts to solve in regard to derivatization/separation in LC-MS orGC-MS.

Methods for detecting amino acids using LC and MS have been reported andinclude, for example, Casetta et al. “Development of a method for rapidquantification of amino acids by liquid chromatography, tandem massspectrometry (LC-MSMS) in plasma” Clin Chem Lab Med (2000) 38: 391-401;Hess et al, “Acid hydrolysis of silk fibroins and determination of theenrichment of isotopically labeled amino acids using precolumnderivatization and high performance liquid chromatography-electrosprayionization mass spectrometry” Anal Biochem (2002) 311:19-26; Ji et al.,“Determination of phenethyl isothiocyanate in human plasma and urine byammonia derivatization and liquid chromatography-tandem massspectrometry” Anal Biochem (2003) 323:39-47; Van Eijik et al.,“Determination of amino acid isotope enrichment using liquidchromatography-mass spectrometry” (1999) Anal Biochem 271:8-17; and Liuet al. “Derivatization of amino acids withN,N-dimethyl-2,4-dinitro-5-fluorobenzylamine for liquidchromatography/electrospray ionization mass spectrometry” (2004) RapidCommun Mass Spectrom 18:1059-65. Improved methods for detecting aminoacids in body fluids are desirable.

SUMMARY OF THE INVENTION

Disclosed are methods for detecting various individual amino acids thatmay be present in a body fluid of an individual. Detection of theindividual amino acids in the body fluid may be used to determinewhether the body fluid contains an abnormal of one or more amino acids.In one aspect, the method involves derivatizing the body fluid aminoacids, separating the derivatized amino acids by liquid chromatography(LC), identifying the derivatized amino acids using mass spectrometry(MS) analysis, and quantifying the derivatized amino acid by comparisonto structurally similar amino acids from a set of amino acids standards.Preferably, the MS is other than tandem MS including, for example,single MS. Structurally similar amino acids share significant structuralcharacteristics such as key functional groups such that theidentification of one amino acid by mass spectrometry can be used toidentify the other structurally similar amino acid in the same method.The set of individual amino acid standards is preferably used as a setof internal standards by adding the set to the body fluid prior toprocessing.

Suitable body fluids include, for example, plasma, serum, saliva, urine,and cerebral spinal fluid (CSF). For methods in which individual aminoacids are detected, identified, and/or quantified in plasma, the levelsmay be compared to the reference ranges provided in Table 1. Plasmaamino acid levels that fall outside of the reference ranges of Table 1may be identified as abnormal. For methods in which individual aminoacids are detected, identified, and/or quantified in urine, the levelsmay be compared to the reference ranges provided in Table 2. Urine aminoacid levels that fall outside of the reference ranges of Table 2 may beidentified as abnormal. For methods in which individual amino acids aredetected, identified, and/or quantified in CSF, the levels may becompared to the reference ranges provided in Table 3. CSF amino acidlevels that fall outside of the reference ranges of Table 3 may beidentified as abnormal. For methods in which individual amino acids aredetected, identified, and/or quantified in saliva, the levels may becompared to the reference ranges provided in Table 4. Saliva amino acidlevels that fall outside of the reference ranges of Table 4 may beidentified as abnormal. In some embodiments, the reference range foreach amino acid varies based on the age of the subject (i.e., neonate,infant, child, or adult).

The set of standards may be non-derivatized and added to the startingbody fluid or to any post processing step prior to derivatization. In apreferred embodiment, the standards comprise individual deuterated aminoacids (i.e., single amino acids containing one or more deuterium ions).The set of standards also may be added to the body fluid amino acidsafter the step of derivatization. In this case, the added standardsshould be derivatized in the same manner as the body fluid amino acids.In one approach, the amino acids are derivatized with an isothiocyanate(e.g., phenyl isothiocynate (PITC). In a preferred embodiment, thederivatizing agent is PITC. Other suitable derivatizing agents mayinclude o-phthaldialdehyde (OPA), 2,4-dinitrofluorobenzene (DNFB), andNα-(2,4-dinitro-5-fluorophenyl)-L-alainamide (FDAA).

The amount of each identified amino acid from a volume of body fluid canbe determined by comparing the signal by MS to the signal of a knownamount of structurally similar amino acid. The amount of the amino acidin the body fluid can then be expressed relative to the volume of bodyfluid analyzed to obtain a concentration of the amino acid in theoriginal body fluid. Quantitative analysis is preferably done withinternal standards.

In some embodiments, the body fluid can be processed to obtain afraction with an enriched concentration of amino acids prior to furtheranalysis. In one approach, a low molecular weight fraction of the bodyfluid is obtained (e.g., by passing the bodily fluid through a molecularweight filter).

In one embodiment, the method may be used to detect at least 20different individual amino acids. In other embodiments, the method maybe used to detect at least 25, 30, 35, or 40 individual amino acids.

For example, the method may be useful for detecting and/or quantifyingany combination of the individual amino acids including, but not limitedto, phosphoserine, sulfo-cysteine, arginosuccinic acid, hydroxyproline,aspartic acid, asparagine, glutamic acid, serine, phosphoethanolamine(PEA), glutamine, glycine, histidine, sarcosine, taurine, carnosine,citrulline, arginine, anserine, 1-methyl-histidine, 3-methyl-histidine,alpha-amino-adipic acid (AAD), threonine, alanine, beta-alanine (BALA),proline, ethanolamine, gamma-amino-butyric acid (GABA),beta-amino-isobutyric acid (BAIA), alpha-amino-butyric acid (AAB),cysteine, tyrosine, valine, methionine, L-allo-cystathionine(cystathionine-A), L-cystathionine (cystathionine-B), cystine,isoleucine, allo-isoleucine, leucine, DL-hydroxylysine (hydroxylysine(1)), DL-allo-hydroxylysine (hydroxylysine (2)), phenylalanine,ornithine, tryptophan, homocystine, arginosuccinic acid (ASA), lysine,and Hawkinsin ((2-L-cystein-S-yl-1,4-dihydroxycyclohex-5-en-1-yl)-aceticacid). In addition, the method may be used to diagnose a disease statebased on the level of any of the detected individual amino acids in bodyfluid.

In one embodiment, the method may be useful for detecting and/orquantifying any combination of the individual amino acids including, butnot limited to, aspartic acid, ASA, sulfo-cysteine, glutamic acid,OH-proline, serine, asparagine, PEA, AAD, glycine, glutamine, sarcosine,histidine, beta-alanine, taurine, citrulline, carnosine, threonine,arginine, anserine, 1-methyl-histidine, 3-methyl-histidine, alanine,GABA, BAIB, proline, ethanolamine, AAB, tyrosine, valine, methionine,cystathionine A, cystathionine B, cystine, isoleucine, allo-isoleucine,leucine, OH-lysine-1, OH-lysine-2, homocystine, phenylalanine,tryptophan, ornithine, and lysine.

In another embodiment, the method may be useful for detecting and/orquantifying any combination of the individual amino acids including, butnot limited to, hydroxyproline, aspartic acid, asparagine, glutamicacid, serine, glutamine, glycine, histidine, sarcosine, taurine,citrulline, arginine, 1,3-methyl-histidine, alpha-amino-adipic acid,threonine, alanine, beta-alanine, proline, ethanolamine,gamma-amino-butyric acid, beta-amino-isobutyric acid,alpha-amino-butyric acid, tyrosine, valine, methionine, L-cystathionine,isoleucine, leucine, phenylalanine, ornithine, tryptophan, homocystine,and lysine.

In yet another embodiment, the method may be useful for detecting and/orquantifying any combination of the individual amino acids including, butnot limited to, hydroxyproline, aspartic acid, asparagine, glutamicacid, serine, glutamine, glycine, histidine, sarcosine, taurine,citrulline, arginine, 1,3-methyl-histidine, alpha-amino-adipic acid,threonine, alanine, beta-alanine, proline, ethanolamine,gamma-amino-butyric acid, beta-amino-isobutyric acid,alpha-amino-butyric acid, tyrosine, valine, methionine, L-cystathionine,isoleucine, leucine, phenylalanine, ornithine, tryptophan, homocystine,lysine, cystine, and hydroxylysine.

In still another embodiment, the method may be useful for detectingand/or quantifying any combination of the individual amino acidsincluding, but not limited to, hydroxyproline, aspartic acid,asparagine, glutamic acid, serine, glutamine, glycine, histidine,sarcosine, taurine, citrulline, arginine, alpha-amino-adipic acid,threonine, alanine, beta-alanine, proline, gamma-amino-butyric acid,beta-amino-isobutyric acid, alpha-amino-butyric acid, tyrosine, valine,methionine, isoleucine, leucine, phenylalanine, ornithine, tryptophan,homocystine, and lysine.

In one embodiment, the method may be useful for detecting and/orquantifying any combination of the individual amino acids including, butnot limited to, phosphoserine, sulfo-cysteine, arginosuccinic acid,hydroxyproline, aspartic acid, phosphoethanolamine, sarcosine,carnosine, anserine, 1,3-methyl-histidine, alpha-amino-adipic acid,beta-alanine, proline, ethanolamine, gamma-amino-butyric acid,beta-amino-isobutyric acid, cysteine, L-allo-cystathionine-A,L-cystathionine, cystine, allo-isoleucine, DL-hydroxylysine,DL-allo-hydroxylysine, and homocystine.

In another embodiment, the method may be useful for detecting and/orquantifying any combination of the individual amino acids including, butnot limited to, phosphoserine, sulfo-cysteine, arginosuccinic acid,hydroxyproline, aspartic acid, phosphoethanolamine, sarcosine,carnosine, anserine, 1,3-methyl-histidine, alpha-amino-adipic acid,beta-alanine, proline, ethanolamine, gamma-amino-butyric acid,beta-amino-isobutyric acid, cysteine, L-allo-cystathionine-A,L-cystathionine, cystine, DL-hydroxylysine, DL-allo-hydroxylysine, andhomocystine. In a further embodiment, the method can be used to identifyany of cysteine, phosphoserine or arginosuccinic acid.

In a further embodiment, the method may be useful for detecting and/orquantifying any combination of the individual amino acids including, butnot limited to, phosphoserine, sulfo-cysteine, arginosuccinic acid,hydroxyproline, phosphoethanolamine, sarcosine, carnosine, anserine,1,3-methyl-histidine, alpha-amino-adipic acid, beta-alanine, proline,ethanolamine, gamma-amino-butyric acid, beta-amino-isobutyric acid,cysteine, L-allo-cystathionine-A, L-cystathionine, cystine,allo-isoleucine, DL-hydroxylysine, DL-allo-hydroxylysine, andhomocystine.

In a further embodiment, the method may be useful for detecting and/orquantifying any combination of the individual amino acids including, butnot limited to, phosphoserine, sulfo-cysteine, arginosuccinic acid,hydroxyproline, phosphoethanolamine, sarcosine, carnosine, anserine,1,3-methyl-histidine, alpha-amino-adipic acid, ethanolamine,gamma-amino-butyric acid, beta-amino-isobutyric acid,L-allo-cystathionine-A, L-cystathionine, cystine, allo-isoleucine,DL-hydroxylysine, DL-allo-hydroxylysine, and homocystine.

In a further embodiment, the method may be useful for detecting and/orquantifying any combination of the individual amino acids including, butnot limited to, phosphoserine, cysteine, arginosuccinic acid,hydroxyproline, phosphoethanolamine, sarcosine, carnosine, anserine,1,3-methyl-histidine, alpha-amino-adipic acid, ethanolamine,gamma-amino-butyric acid, beta-amino-isobutyric acid,L-allo-cystathionine-A, L-cystathionine, cystine, DL-hydroxylysine,DL-allo-hydroxylysine, and homocystine.

As used herein, “sulfo-cysteine” is a short-hand representationrecognized in the art for a single amino acid, L-cysteine-S-sulfate,which may also be referred to as S-sulfo-L-cysteine, S-sulfo-cysteine,S-sulfocysteine, and sulfocysteine. Sulfo-cysteine has the structureCyS-SO₃H. See e.g., Abbas, et al., S-Sulfo-Cysteine is an EndogenousAmino Acid in Neonatal Rat Brain but an Unlikely Mediator of CysteineNeurotoxicity, Neurochemical Research (2008), 33:301-307 (which uses theterm S-sulfo-cysteine); Coloso, et al., Metabolism of Cyst(e)ine in RatEnterocytes, J. Nutrition (1989), 119:1914-1924 (which uses the termsulfocysteine throughout the article); Henderson, Amino Acid Pot Pourri,Department of Clinical Biology at St. James's University Hospital (whichuses the terms S-sulfocysteine (for example on p. 16), sulfocysteine (onp. 18 and 20), and sulfocys (on p. 19) to indicate the same amino acid);Parvy, et al., A New Pitfall in Plasma Amino Acid Analysis, ClinicalChem. (1989), p. 178 (which uses the term sulfocysteine); Description ofService Schedule and Guidelines for Sample Shipment, Medical GeneticsProgram, Duke University Medical Center (which indicates a test isavailable for sulfocysteine in urine, p. 4); The Biochrom 30 Amino AcidSystem product brochure (which indicates on p. 9 that the instrument maybe used to measure sulfocysteine); and the Annual Report ERNDIM-EQAS2007 (which indicates on p. 2 that sulphocysteine is available forpurchase from Sigma-Aldrich under catalog number C2196 (which is thecatalog number for L-Cysteine S-Sulfate)).

In other embodiments, the method quantifies at least two, at leastthree, at least four, at least five, at least seven, at least ten, atleast fifteen, at least twenty, at least twenty five, or at least thirtyor more individual amino acids.

The disclosed methods may be used as a basis for diagnosis, or formonitoring the effectiveness of treatment for a variety of diseasesknown to be associated with abnormal levels of individual amino acid(i.e., single amino acids apart from dipeptides and polypeptides). Forexample, the levels of leucine, isoleucine, valine, lysine, and/orhistidine may be used to diagnose and/or monitor kidney failure; cystineand/or glutamine may be used to diagnose and/or monitor Crohn's disease,ulcerative colitis, and/or chronic fatigue syndrome; tryptophan and/orhistidine may be used to diagnose and/or monitor Wilson's disease;alanine may be used to diagnose and/or monitor Cushing's disease orgout; valine, leucine, and/or isoleucine may be used to diagnose and/ormonitor diabetes and/or Maple Syrup Urine Disease; tyrosine and/orphenylalanine may be used to diagnose and/or monitor hyperactivitydisorders; and hawkinsin may be used to diagnose and/or monitorhawkinsinuria.

LC separation of derivatized amino acids may be performed using any typeof LC system such as are commercially available. A suitable LC column isone that has a packing material which includes minute particles (e.g.,silica particles having a diameter of about 2-5 μm, and preferably about3 μm). The particles typically have pores of about 50 to 300 angstroms,and preferably about 150 angstroms. The particles typically have asurface area of about 50-600 m²/g, and preferably about 100 m²/g.

The particles may include a hydrophobic stationery phase bonded to theirsurface. In one embodiment, the hydrophobic stationery phase may be analkyl phase, which may include C-4, C-8, and C-18 (preferably C-18).

The column may have any suitable dimensions. Preferably, the column hasa diameter of about 0.5 mm to about 5 mm and a length of about 15 mm toabout 300 mm, and most preferably, a diameter of about 2 mm and a lengthof about 50 mm.

LC separation of derivatized amino acids may also be performed using ahydrophobic solvent or a solvent mixture that includes a hydrophobicsolvent in a gradient as a mobile phase to elute the amino acids. In oneembodiment, the derivatized fraction may be applied to the column in anaqueous buffer (i.e., a hydrophilic solvent) and the amino acids may beeluted by applying a mobile phase to the column that has an increasingamount of organic solvent (i.e., a hydrophobic solvent). For example,the aqueous buffer may include (95% H₂O, 5% acetonitrile), and the aminoacids may be eluted from the column by gradually increasing theconcentration of acetonitrile to about 100% in the mobile phase. Ifdesirable, the mobile phase may be heated to a temperature of about40-60° C., preferably about 50° C. In addition, the mobile phase mayoptionally include one or more additional reagents that are usefulduring LC and/or MS. For example, ammonium acetate or acetic acid.

MS analysis of derivatized amino acids may be performed by ionization ofthe sample. Suitable ionization techniques include electrosprayionization (ESI), atmospheric pressure chemical ionization (APCI),photoinonization, electron ionization, fast atom bombardment(FAB)/liquid secondary ionization (LSIMS), matrix assisted laserdesorption ionization (MALDI), field ionization, field desorption,thermospray/plasmaspray ionization, and particle beam ionization.Preferably, MS is performed using ESI. Further, MS may be performedusing a negative or positive ion mode, and preferably a negative ionmode.

MS analysis of derivatized amino acids may be performed with any ofseveral types of ion analyzers including quadrupole analyzers, ion trapsanalyzers, and time-of-flight analyzers. Preferably, MS may be performedusing a quadrupole analyzer. The ions generated during MS may bedetected by using several detection modes including selective ionmonitoring mode (SIM) and scanning mode. Preferably, the ions aredetected by using SIM. MS is preferably other than tandem MS.

Also provided is a method of diagnosing the existence of a metabolicdisorder involving amino acid metabolism in an individual. The methodcomprises determining if a body fluid contains an abnormal level of oneor more amino acids by first (a) derivatizing the body fluid aminoacids; (b) separating the derivatized amino acids by liquidchromatography (LC); (c) subjecting the separated derivatized aminoacids to mass spectroscopic analysis (MS) using a mass spectrometer; and(d) using the MS analysis to identify the amount of derivatized aminoacids from the body fluid by comparing to structurally similar aminoacids from a set of amino acid standards. Various embodiments of thismethod are similar to those already discussed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a table showing various amino acids detected and quantifyingby the methods described herein. Third column indicates MW “molecularweight”; fourth column indicates PITC molecular weight; fifth columnindicates molecular weight of each PITC derivatized amino acids; sixcolumn is the LC retention time; seventh column indicates massspectrometry ions observed.

FIG. 2 shows the results of an MS analysis of a single sample containingthe indicated amino acids, previously subject to LC. The ion sizecharacteristic of each amino acid is indicated.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used herein, “amino acid” means any molecule that includes analpha-carbon atom covalently bonded to an amino group and an acid group.The acid group may include a carboxyl group. “Amino acid” may includemolecules having one of the formulas:

wherein R is a side group and Z includes at least 3 carbon atoms. “Aminoacid” includes, but is not limited to, the twenty endogenous human aminoacids and their derivatives such as lysine, asparagine, threonine,serine, isoleucine, methionine, proline, histidine, glutamine, arginine,glycine, aspartic acid, glutamic acid, alanine, valine, phenylalanine,leucine, tyrosine, cysteine, tryptophan, phosphoserine (PSER),sulfo-cysteine, arginosuccinic acid (ASA), hydroxyproline,phosphoethanolamine (PEA), sarcosine (SARC), taurine (TAU), carnosine(CARN), citrulline (CIT), anserine (ANS), 1,3-methyl-histidine (ME-HIS),alpha-amino-adipic acid (AAA), beta-alanine (BALA), ethanolamine (ETN),gamma-amino-butyric acid (GABA), beta-amino-isobutyric acid (BAIA),alpha-amino-butyric acid (BABA), L-allo-cystathionine (cystathionine-A;CYSTA-A), L-cystathionine (cystathionine-B; CYSTA-B), cystine,allo-isoleucine (ALLO-ILE), DL-hydroxylysine (hydroxylysine (1)),DL-allo-hydroxylysine (hydroxylysine (2)), ornithine (ORN), homocystine(HCY), and derivatives thereof. “Amino acids” also includesstereoisomers such as the D-amino acid and L-amino acid forms. Unlessspecifically indicated otherwise, the term “amino acid” refers to theindividual (i.e., free) amino acid molecules apart from amino acidspresent in dipeptides, polypeptides, and proteins.

As used herein, “body fluid” means any fluid that can be isolated fromthe body of an individual. For example, “body fluid” may include blood,plasma, serum, bile, saliva, urine, tears, perspiration, cerebrospinalfluid (CSF), and the like. Preferably the body fluid is plasma, serum,cerebrospinal fluid, urine, or saliva, with plasma being the mostpreferred.

As used herein, “derivatizing” means reacting two molecules to form anew molecule. For example, an amino acid may be derivatized by reactingthe amino acid with a derivatizing agent to form a derivatized aminoacid. Derivatizing may include reacting the alpha amino group of theamino acid with an electrophilic atom of a derivatizing agent to form acovalent bond. Derivatizing agents may include isothiocyanate groups,dinitro-fluorophenyl groups, nitrophenoxycarbonyl groups, and/orphthalaldehyde groups.

As used herein, “liquid chromatography” (LC) means a process ofselective retardation of one or more components of a fluid solution asthe fluid uniformly percolates through a column of a finely dividedsubstance, or through capillary passageways. The retardation resultsfrom the distribution of the components of the mixture between one ormore stationery phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves countercurrent to the stationery phases. The process is usedfor analysis and separation of mixtures of two or more substances.“Liquid chromatography” includes reverse phase liquid chromatography(RPLC) and high pressure liquid chromatography (HPLC).

As used herein, “mass spectroscopic” analysis or “mass spectrometry” (MSanalysis) means an analytical technique to identify unknown compoundsincluding: (1) ionizing the compounds and potentially fractionating thecompounds to form charged compounds; and (2) detecting the molecularweight of the charged compound and calculating a mass-to-charge ratio(m/z). The compound may be ionized and detected by any suitable means. A“mass spectrometer” includes means for ionizing compounds and detectingcharged compounds.

As used herein, “electrospray ionization” means a technique used in massspectrometry to ionize macromolecules and to overcome the propensity ofmacromolecules to fragment. In “electrospray ionization” a liquid ispushed through a very small charged metal capillary by a carrier gas.The liquid contains the substance which is to be studied, the analyte,as well as a large amount of solvent, which is usually much morevolatile then the analyte. The charge contained in the capillarytransfers to the liquid which charges the analyte molecule. As likecharges repel, the liquid pushes itself out of the capillary and forms amist or an aerosol of small droplets about 10 μm across, to increase thedistance between the similarly charged molecules. A neutral carrier gasis used to evaporate the neutral solvent in the small droplets, this inturn brings the charged analyte molecules closer together. The proximityof the molecules becomes unstable, however, and as the similarly chargesmolecules come closer together, the droplets once again explode. Thisprocess repeats itself until the analyte is free of solvent and a loneion is formed. The lone ion is transported to a mass analyzer.

As used herein, a “quadrupole analyzer” is a mass analyzer composed ofquads (i.e., two pairs of metallic rods aligned in parallel), whereinone pair of rods is at a positive electrical potential and the other setof rods is at a negative potential. To be detected, an ion must passthrough the center of a trajectory path bordered and parallel to thealigned rods. When the quads are operated at a given amplitude of directcurrent and radio frequency voltages, only ions of a given m/z ratiowill resonate and have a stable trajectory to pass through thequadrupole and be detected. “Positive ion mode” means a mode whereinpositively charged ions are detected by the mass analyzer. “Negative ionmode” means a mode wherein negatively charged ions are detected by themass analyzer. For “single ion monitoring” or “selected ion monitoring”(i.e., SIM), the amplitude of the direct current and the radio frequencyvoltages are set to observe only a specific mass.

As used herein, a “low molecular weight fraction” is a fraction that isenriched in one or more low molecular weight molecules. A low molecularweight molecule typically has a molecular weight of less than about 1000daltons, and more typically less than about 500 daltons.

As used herein, “hydrophobic” means not dissolving or dissolving poorlyin water. “Hydrophobic” compounds include long chain alkanes. Ahydrophobic solvent is a solvent that is capable of dissolving ahydrophobic compound.

As used herein “about” when used in the context of a number means thenumber plus or minus 10%.

Disclosed is a method for identifying and/or quantifying amino acids ina body fluid. The body fluid can be blood, plasma, serum, bile, saliva,urine, cerebrospinal fluid, and the like. A preferred body fluids areplasma, serum, CSF, urine, or saliva, with plasma being the mostpreferred.

A set of individual amino acid standards representing the types of aminoacids that might be present in a particular body fluid is preferablyadded to the body fluid sample before any processing. The set of aminoacid standards preferably contains a known amount of each individualamino acid present in the set. A set of amino acid standards may includeone or more amino acids from the group consisting of lysine, asparagine,threonine, serine, isoleucine, methionine, proline, histidine,glutamine, arginine, glycine, aspartic acid, glutamic acid, alanine,valine, phenylalanine, leucine, tyrosine, cysteine, tryptophan,phosphoserine, sulfo-cysteine, arginosuccinic acid, hydroxyproline,phosphoethanolamine, sarcosine, taurine, carnosine, citrulline,anserine, 1,3-methyl-histidine, alpha-amino-adipic acid, beta-alanine,ethanolamine, gamma-amino-butyric acid, beta-amino-isobutyric acid,alpha-amino-butyric acid, L-allo-cystathionine (cystathionine-A),L-cystathionine (cystathionine-B), cystine, allo-isoleucine, leucine,DL-hydroxylysine (hydroxylysine (1)), DL-allo-hydroxylysine(hydroxylysine (2)), ornithine, tryptophan, homocystine, and isomersthereof (e.g., stereoisomers).

The amino acids of the set of amino acid standards may be modified sothat they can be easily discriminated from the corresponding amino acidsfound in the body fluid. The internal standard amino acid preferablybehaves closest to the amino acid that it is chosen to representchemically and physically but fragments to ions of a different mass uponmass spectrometric analysis. Thus, a preferred set of amino acidsstandards is deuterated.

Body fluid may be processed prior to derivatization to obtain anenriched preparation of the amino acids. Various procedures may be usedfor this purpose depending on the type of body fluid. These includefiltration, precipitation, centrifugation, combinations thereof and thelike. Separation of a low molecular weight fraction is a preferredapproach. Size separation on small volumes of sample is preferablyperformed by filtering using a low molecular weight cutoff filter. Thefiltered body fluid sample (i.e., permeate) will include free aminoacids and the retained components (i.e., retentate) will include highmolecular weight components such as proteins. Suitable filters forgenerating a filtrate include 45 micron, 22 micron and 100,000, 50,000and 10,000 dalton cutoff filters. In addition, high molecular weightcomponents may be precipitated from the plasma sample by adding alcohol(e.g., methanol) or acid to the sample. High molecular weight componentsmay also be removed from the sample by high speed centrifugation.

Derivatization of amino acids is performed following any necessaryprocessing of the body fluid sample. The amino acids in the sampletypically are derivatized to facilitate separation and/or detection offree amino acids in the sample during LC-MS (e.g., pre-columnderivatization is first performed where LC is subsequently performed).The derivatizing agent may include substituents that facilitatedetection of the derivatized amino acids during or after chromatography(e.g., fluorophores or chromophores). In addition, the derivatizingagent may include substituents that facilitate ionization of thederivatized amino acids during mass spectrometry. Typical derivatizingagents include isothiocyanates (e.g., phenyl isothiocynate (PITC)),o-phthaldialdehyde (OPA), 2,4-dinitrofluorobenzene (DNFB), andNα-(2,4-dinitro-5-fluorophenyl)-L-alainamide (FDAA). In a preferredembodiment, the derivatizing agent is PITC.

After the amino acids in the sample have been derivatized, the sample issubjected to chromatographic separation, preferably high pressure liquidchromatographic separation, and mass spectrometry (i.e., LC-MS).

Liquid chromatography and mass spectrometry may be performed by placingthe derivatized sample in an instrument that includes a chromatographiccolumn in flow communication with a mass spectrometer. Thechromatographic column typically includes a medium (i.e., a packingmaterial) to facilitate separation of the derivatized amino acids (i.e.,fractionation). The medium may include minute particles that have adiameter of approximately 2-6 μm, preferably about 3 μm. For example,the particles may be silica particles. The particles may have pores thathave a diameter of approximately 50-300 angstroms, preferably 150angstroms. Additionally, the particles may have a surface area ofapproximately 50-600 m²/g, preferably 100 m²/g.

The particles include a bonded surface that interacts with thederivatized amino acids to facilitate separation of the amino acids. Onesuitable bonded surface is a hydrophobic bonded surface such as an alkylbonded surface. Alkyl bonded surfaces may include C-4, C-8, or C-18bonded alkyl groups, preferably C-18 bonded groups.

The column may have any suitable dimensions. In particular, the columnmay have a diameter of about 0.5-5 mm and a length of about 15-300 mm.Preferably, the column has a diameter of about 2 mm and length of about50 mm.

Suitable media for preparing a chromatographic column and/or preparedcolumns may be obtained from commercial sources. In particular, suitablecolumns may be obtained from Thermo Electron Corporation (e.g., 250×2.1mm, 5 μm, BetaBasic C18 column).

The chromatographic column includes an inlet port for receiving a sampleand an outlet port for discharging an effluent that includes thefractionated sample. In the method, the derivatized sample is applied tothe column at the inlet port, eluted with a solvent or solvent mixture,and discharged at the outlet port. Different solvent modes may beselected for eluting the amino acids. For example, liquid chromatographymay be performed using a gradient mode, an isocratic mode, or apolytyptic (i.e. mixed) mode. Preferably, liquid chromatography isperformed using a gradient mode. In the gradient mode, the derivatizedsample is applied to the column and a mixture of two solvents (i.e., themobile phase) is passed through the column to elute the amino acids.Generally, as known in the art, one of the solvents will tend to berelatively hydrophilic, and the other solvent will tend to be relativelyhydrophobic. As a specific example of a solvent combination found to besuitable in the practice of the present method, the hydrophilic solventmay be 95% H₂O, 5% acetonitrile and the hydrophobic solvent may be 100%acetonitrile. Optionally, the solvent combination may include one ormore reagents to facilitate separation and/or detection of thederivatized amino acids (e.g., 20 mM ammonium acetate). Some reagentsmay be added to the mobile phase to improve the shape of thechromatographic peak and/or to provide a source of ions for LC-MS.

In most cases, to perform liquid chromatography with a gradient solvent,two pumps are used that mix the two solvents. Initially, as the solventsare mixed, the solvent mixture that is passed through the column (i.e.,mobile phase) includes mostly hydrophilic solvent. Gradually, the amountof hydrophilic solvent in the mixture is decreased and the amount ofhydrophobic solvent in the mixture is increased to create a solventgradient. Ultimately, the solvent mixture that is passed through thecolumn includes mostly hydrophobic solvent. In this manner, hydrophilicamino acids will be eluted before hydrophobic amino acids.

The mass spectrometer includes an inlet port for receiving thefractionated sample that is in flow communication with the outlet portof the chromatographic column. The mass spectrometer is capable ofgenerating one or more mass spectroscopic data sets for identifying oneor more amino acids in the sample. Suitable instruments for performingLC-MS may be obtained from commercial sources. In particular, suitableinstruments for performing LC-MS may be obtained from AgilentTechnologies (e.g., Agilent 1100 Series LC/MSD).

The mass spectrometer will include an ion source for ionizing thefractionated sample and creating charged molecules for further analysis.Ionization of the sample may be performed by electrospray ionization(ESI), atmospheric pressure chemical ionization (ACPI),photoinonization, electron ionization, fast atom bombardment(FAB)/liquid secondary ionization (LSIMS), matrix assisted laserdesorption ionization (MALDI), field ionization, field desorption,thermospray/plasmaspray ionization, and particle beam ionization.Electrospray ionization is preferred.

After the sample has been ionized, the positively charged or negativelycharged ions thereby created may be analyzed to determine amass-to-charge ratio (i.e., m/z). Preferably, the negatively chargedions are analyzed. Suitable analyzers for determining mass-to-chargeratios include quadropole analyzers, ion traps analyzers, andtime-of-flight analyzers. Preferably, the mass-to-charge ratio isdetermined using a quadropole analyzer. The ions may be detected byusing several detection modes. For example, selected ions may bedetected (i.e., using a selective ion monitoring mode (SIM)), oralternatively, ions may be detected using a scanning mode. Preferably,the ions are detected by using SIM.

Example 1—Analytical Procedures

Urine, plasma, and cerebrospinal fluid (CSF) samples were obtained fromnormal individuals. Heparinized plasma samples were collected frompatients after an overnight fast. Non-fasting samples for pediatricpatients were used. The following procedure was used for amino acidquantification in urine, plasma, and CSF.

Deuterated internal standards were added to about 100 μl of the testmaterial (i.e., plasma, urine, or CSF) to form a test mixture. The testmixture then was passed through a 10,000 molecular weight filter toprovide a low molecular weight filtrate fraction as a test sample andthen dried under nitrogen at 40-75° C. The dried sample was dissolved inabout 25 μl of a redry solution (equal volumes of methanol, 1M sodiumacetate, and triethylamine) and dried under nitrogen at 40° C. Thesample was dissolved in 50 μl of a derivatizing solution (1.12 μl of100% methanol, 1.60 μl water, 1.60 μl of 100% triethylamine, and 1.60 μlof 100 phenylisothiocyanate (PITC)). The dissolved sample was thenheated at about 40° C. for about 15-20 minutes and then dried underliquid nitrogen at 50-60° C. The dried sample was dissolved in 100 μl ofa reconstitution solution (95% H₂O, 5% acetonitrile), vortexed, andtransferred to vials for LC/MS.

LC/MS determination of the sample amino acids was performed using anAgilent 1100 Series LC/MSD with a Thermo Beta-Basic C-18 (250×2.1 mm)HPLC column. The mobile phase consisted of (A) 20 mM ammonium acetateand (B) 100% acetonitrile, heated to 50° C. Sample amino acids wereeluted from the column using a step-gradient as follows:

Maximum Time Mobile Phase Flow Pressure Step (minutes) % B (mL/Minute)(bar) 1 0.00 97.0 0.550 350 2 1.40 97.0 0.550 350 3 4.50 92.0 0.550 3504 6.50 90.0 0.550 350 5 10.50 82.0 0.550 350 6 13.50 80.0 0.650 350 716.50 73.0 0.650 350 8 17.50 55.0 0.650 350 9 18.50 50.0 0.650 350 1019.70 20.0 0.550 350 11 20.70 20.0 0.550 350 12 21.00 97.0 0.550 350

As the separated amino acids exit the HPLC column, they were introducedinto the spray chamber where the effluent is sprayed and de-solvated bythe electrospray ion source. The PITC derivatives were negativelycharged during the electrospray process and then further separatedthrough the quadrupole mass filter. Amino acid ions were detected andtheir abundances measured. The ratio of the area abundances of eachamino acid ion to its internal standard was plotted against a six-pointcalibration curve. FIGS. 1 and 2 show the results of a typical run.

Example 2—Quantification of Amino Acid Content in Plasma

Amino acid analysis was performed on heparinized plasma samples obtainedfrom neonates (≤30 days), infants (1-23.9 months), children (2-17.9years), and adults 18 years). All individuals were assessed asclinically normal or obtained from samples submitted for infectiousdisease determinations. All subjects were fully ambulatory, communitydwelling, healthy, and on no medications. The demographics of the testgroups is as follows:

# of subjects # of # of Test Group (n) males females Neonates 55 33 22(≤30 days) Infants 115 59 56 (1-23.9 months) Children 134 76 58 (2-17.9years) Adults 134 64 70 (≥18 years

Based on the analysis of these plasma samples, normal Reference Rangeswere constructed using standard parametric and non-parametricstatistical procedures. If the data was determined to be Gaussian, theappropriate mean±2 SD range was chosen. If the data was non-gaussian,the non-parametric 95 percentile range or observed range was chosen.Table 1 provides normal reference ranges for each amino acid assayed,along with the limit of quantification (LOQ). Reference ranges areprovided in μM (micromoles per liter). The normal range for amino acidswhich are undetectable should be taken to be less than or equal to theLOQ.

TABLE 1 Adult and Pediatric Amino Acid Levels in Plasma Amino Acid LOQNeonates Infants Children Adults Pserine N/A  ≤0.67  ≤0.56 Undetectable ≤0.90 Aspartic Acid 1.0 2.4-19.5 2.3-14.3 1.3-8.2  0.9-3.9Sulfo-Cysteine UNK 0.13-1.60   ≤1.66  ≤1.62 0.44-3.95 Glutamic Acid 1.051-277 32-185  9-109 10-97 Arginosuccinic Acid 1.0  ≤1.15  ≤1.10  ≤1.00 ≤1.00 (ASA) Hydroxyproline 1.0 13-72  7-63 6-32  4-27 (OH-Proline)Hawkinsin 1.0 Undetectable Undetectable Undetectable Undetectable SerineUNK 87-241 83-212 85-185  65-138 Asparagine 1.0 12-70  20-77  23-70 31-64 α-Amino Adipic 1.0 ≤2.8 ≤3.6 ≤2.1 ≤2.4 Acid (AAD) Glycine 4.0133-409  103-386  138-349  122-322 Glutamine UNK 240-1194 303-1459405-923  428-747 Sarcosine 1.0 ≤4.5 ≤4.0 ≤3.9 ≤3.7 Phospho- 2.0Undetectable Undetectable Undetectable Undetectable Ethanolamine (PEA)β-Alanine 2.0 ≤8.3 ≤7.8 ≤4.7 ≤4.8 (BALA) Taurine UNK 29-161 26-13032-114  31-102 Histidine UNK 40-143 42-125 54-113  60-109 Citrulline 1.03-35 4-50 9-52 16-51 Carnosine 1.0 ≤10.0  ≤6.1 Undetectable UndetectableArginine UNK 14-135 30-147 38-122  43-407 Threonine UNK 56-392 40-42859-195  67-198 Alanine UNK 83-447 119-523  157-481  200-4831-Methylhistidine 1.0 ≤4.2 ≤8.6 ≤27.4  ≤47.1  (1-Me-His) Anserine 1.0Undetectable Undetectable Undetectable Undetectable γ-Amino-Butyric 1.0Undetectable Undetectable ≤2.2 ≤3.1 Acid (GABA) 3-Methylhistidine 1.0≤9.9 ≤8.2 1.4-6.3  2.1-8.6 (3-Me-His) β-Amino-Isobutyric 3.0 ≤9   ≤8  ≤6   Undetectable Acid (BAIB) Proline 1.0 87-375 104-348  99-351 104-383Ethanolamine UNK  8-106 5-19 5-15  5-13 α-Amino-Butyric 1.0 1-20 4-306-30  7-32 Acid (AAB) Cysteine 1.0 ≤5.3 ≤16.3  ≤34.6   6.6-73.5 TyrosineUNK 33-160 24-125 31-108 38-96 Valine UNK 57-250 84-354 130-307  132-313Methionine 1.0 13-45  12-50  14-37  16-34 Cystathionine-A 1.0Undetectable Undetectable Undetectable Undetectable (Cysta-A)Cystathionine-B 1.0 Undetectable Undetectable Undetectable Undetectable(Cysta-B) Cystine 1.0 ≤25   ≤19   ≤39    8-52 Isoleucine 1.0 12-92 10-109 33-97  34-98 Allo-Isoleucine UNK Undetectable UndetectableUndetectable Undetectable (allo-Ile) Leucine UNK 23-172 43-181 65-179 73-182 Hydroxy-Lysine-1 1.0 3-10 1-8  1-4  1-7 (OH-Lysine-1)Hydroxy-Lysine-2 1.0 Undetectable Undetectable Undetectable 2.34 or less(OH-Lysine-2) Homocystine 1.0 Undetectable Undetectable UndetectableUndetectable Phenylalanine UNK 30-79  31-92  38-86  40-74 Tryptophan 1.017-85  16-92  30-94  40-91 Ornithine 2.0 29-168 19-139 33-103 27-83Lysine UNK 66-226 70-258 98-231 119-233

Example 3—Quantification of Amino Acid Content in Urine

Amino acid analysis was performed on urine samples obtained fromneonates (≤30 days), infants (1-23.9 months), children (2-17 years), andadults (>17 years). All individuals were assessed as clinically normalor obtained from samples submitted for infectious diseasedeterminations. All subjects were fully ambulatory, community dwelling,healthy, and on no medications. The demographics of the test groups isas follows:

# of subjects # of # of Test Group (n) males females Neonates 17 14 3(≤30 days) Infants 40 23 17 (1-23.9 months) Children 80 42 38 (2-17years) Adults 111 55 56 (>17 years)

The amino acid content of urine is initially measured on a concentration(μmol amino acid per liter of urine; μM) basis. The amino acidconcentrations were then normalized based on the urinary creatininelevels and normal Reference Ranges were constructed using standardparametric and non-parametric statistical procedures. If the data wasdetermined to be Gaussian, the appropriate mean±2 SD range was chosen.If the data was non-gaussian, the non-parametric 95 percentile range orobserved range was chosen. Table 2 provides normal reference ranges(mmol/mol creatinine) for each amino acid assayed. The limit ofquantification (LOQ) is based on the amino acid determination in urine,unadjusted for creatinine. The normal range for amino acids which areundetectable should be taken to be less than or equal to the LOQ.

TABLE 2 Adult and Pediatric Amino Acid Levels in Urine (mmol/molcreatinine) LOQ Amino Acid (μM) Neonates Infants Children Adults Pserine1  ≤8  ≤3 Undetectable ≤247  Aspartic Acid 1  ≤7 ≤11 ≤2 ≤2Sulfo-Cysteine 1 ≤50 ≤45 ≤30  ≤11  Glutamic Acid 1 4-19 3-30 ≤10  ≤3Arginosuccinic Acid 1 ≤15  ≤9 ≤6 ≤4 (ASA) Hydroxyproline 1 30-485  2-345≤4 ≤2 (OH-Proline) Hawkinsin 1 Undetectable Undetectable UndetectableUndetectable Serine 1 44-454 39-422 13-127 10-71  Asparagine 1 8-42 5-132 3-42 2-37 α-Amino Adipic UNK ≤10 ≤36 ≤34  ≤11  Acid (AAD) Glycine1 215-2053 105-413  23-413 ≤330  Glutamine UNK ≤355  41-396 18-18821-182 Sarcosine 1 ≤18 ≤19 ≤2 ≤69  Phospho- 2 ≤23 ≤32 ≤15  ≤4Ethanolamine (PEA) β-Alanine 2  ≤9 ≤15 ≤5 ≤10  (BALA) Taurine 1 ≤650 ≤670  ≤255  ≤232  Histidine 1 40-301 56-543  9-425 17-266 Citrulline 1 ≤4 ≤13 ≤4 ≤2 Carnosine 1 9-72 15-65  ≤23  ≤8 Arginine 2 ≤30 ≤35 ≤8 ≤5Threonine 1 ≤112   9-158 4-60 4-46 Alanine UNK 45-264 16-294  8-156 9-671-Methylhistidine 1 ≤16 4-71  5-400 ≤204  (1-Me-His) Anserine 1  ≤8 ≤30≤87  ≤411  γ-Amino-Butyric 1   ≤1.4   ≤1.5   ≤1.6   ≤1.6 Acid (GABA)3-Methylhistidine UNK 9-45 14-35  11-40  10-35  (3-Me-His)β-Amino-Isobutyric 3 ≤269  ≤309  ≤133  ≤88  Acid (BAIB) Proline 1 ≤219 ≤216  ≤11  ≤2 Ethanolamine 1 87-490 54-176 27-114 21-65  α-Amino-Butyric1  ≤7  ≤7 ≤5 ≤2 Acid (AAB) Cysteine 1 Undetectable Undetectable ≤4 ≤5Tyrosine UNK 4-59 10-69  3-48 3-19 Valine 2 2-20 4-21 2-20 2-5 Methionine 1  ≤7  ≤7 ≤5 ≤2 Cystathionine-A 1  ≤2  ≤1 ≤2 ≤3 (Cysta-A)Cystathionine-B 1 ≤20 ≤29 ≤8 ≤5 (Cysta-B) Cystine UNK 15-58  6-28 3-203-13 Isoleucine 3  ≤9 ≤12 ≤5 ≤3 Allo-Isoleucine 1 UndetectableUndetectable Undetectable Undetectable (allo-Ile) Leucine 2 ≤23 ≤24 ≤13 ≤6 Hydroxy-Lysine-1 1 ≤83 ≤71 ≤8 ≤8 (OH-Lysine-1) Hydroxy-Lysine-2 1  ≤3 ≤3 Undetectable Undetectable (OH-Lysine-2) Homocystine 1 Undetectable ≤4 Undetectable Undetectable Phenylalanine UNK 3-24 6-39 2-22 2-9 Tryptophan 1 2-21 5-46 2-27 2-14 Ornithine 2 ≤39 ≤11 ≤5 ≤4 Lysine UNK13-284  4-239  3-112 3-59 Hydroxy-Lysine, 1  5-117 2-72 ≤8 ≤8 TotalCystathionine, Total 1 2-20 ≤29 ≤8 ≤9

Example 4—Quantification of Amino Acid Content in Cerebrospinal Fluid(CSF)

Amino acid analysis was performed on CSF samples obtained from neonates(≤3 months), infants (3-23.9 months), children (2-10 years), and adults(>10 years). All individuals were assessed as clinically normal orobtained from samples submitted for infectious disease determinations.All subjects were fully ambulatory, community dwelling, healthy, and onno medications. The demographics of the test groups is as follows:

# of subjects # of # of Test Group (n) males females Neonates 27 23 4(≤3 months) Infants 22 16 6 (3-23.9 months) Children 21 9 12 (2-10years) Adults 57 21 36 (>10 years)

Based on the analysis of these CSF samples, normal Reference Ranges wereconstructed using standard parametric and non-parametric statisticalprocedures. If the data was determined to be Gaussian, the appropriatemean±2 SD range was chosen. If the data was non-gaussian, thenon-parametric 95 percentile range or observed range was chosen. Table 3provides normal reference ranges for each amino acid assayed, along withthe limit of quantification (LOQ). Reference ranges are provided in μM(micromoles per liter). The normal range for amino acids which areundetectable should be taken to be less than or equal to the LOQ.

TABLE 3 Adult and Pediatric Amino Acid Levels in CSF Amino Acid LOQNeonates Infants Children Adults Pserine N/A  ≤4.62  ≤2.39  ≤3.85  ≤4.19Aspartic Acid 1.0 ≤2.7 Undetectable Undetectable ≤2.0 Sulfo-Cysteine UNK0-1  0-1  0-1  0-1  Glutamic Acid 1.0 1-9  ≤5.1 ≤10.6  1.1-13.2Arginosuccinic Acid 1.0 ≤4.3 ≤2.4 ≤3.0 ≤2.5 (ASA) Hydroxyproline 1.00.9-3.9  ≤1.6 Undetectable ≤1.7 (OH-Proline) Hawkinsin 1.0 UndetectableUndetectable Undetectable Undetectable Serine 30-88  22-61  15-62  9-41Asparagine 1.0 ≤27   ≤13   ≤25   ≤24   α-Amino Adipic 1.0 UndetectableUndetectable Undetectable Undetectable Acid (AAD) Glycine 4.0 3-26 ≤12  ≤13   ≤10   Glutamine UNK 525-1583 386-742  377-1738 361-1175 Sarcosine1.0 Undetectable Undetectable Undetectable Undetectable Phospho- 2.0Undetectable Undetectable ≤4.2 ≤4.8 Ethanolamine (PEA) β-Alanine 2.0Undetectable Undetectable Undetectable Undetectable (BALA) Taurine UNK0-18 ≤8   1-8 1-8  Histidine UNK 8-32 4-25 7-25 7-22 Citrulline 1.0 1-4 ≤3   1-2 ≤2   Carnosine 1.0 Undetectable Undetectable UndetectableUndetectable Arginine UNK 2-27 7-32 9-31 10-32  Threonine UNK 23-10410-55  8-85 12-64  Alanine UNK 13-50  8-48 5-62  1-107 1-Methylhistidine1.0 ≤6.4 ≤9.0 ≤3.8 ≤4.2 (1-Me-His) Anserine 1.0 ≤26   ≤27   ≤19   ≤28  γ-Amino-Butyric 1.0 Undetectable Undetectable ≤2.2 ≤3.1 Acid (GABA)3-Methylhistidine 1.0 ≤3.3 ≤1.8 ≤2.5 ≤2.7 (3-Me-His) β-Amino-Isobutyric3.0 Undetectable Undetectable Undetectable Undetectable Acid (BAIB)Proline 1.0 ≤3.9 ≤2.3 ≤1.7 ≤5.9 Ethanolamine UNK 1-46 3-19 5-40 4-23α-Amino-Butyric 1.0 ≤6   ≤6   1-11 1-11 Acid (AAB) Cysteine 1.0Undetectable Undetectable Undetectable Undetectable Tyrosine UNK 9-415-20 5-32 5-18 Valine UNK 11-31  8-19 2-37 7-42 Methionine 1.0 2-14 1-7 ≤9   1-8  Cystathionine-A 1.0 Undetectable Undetectable UndetectableUndetectable (Cysta-A) Cystathionine-B 1.0 Undetectable UndetectableUndetectable Undetectable (Cysta-B) Cystine 1.0 ≤3.7 ≤3.2 ≤1.6 ≤2.2Isoleucine 1.0 3-11 3-7  2-13 3-10 Allo-Isoleucine UNK UndetectableUndetectable Undetectable Undetectable (allo-Ile) Leucine UNK 7-22 7-128-27 9-32 Hydroxy-Lysine-1 1.0 Undetectable Undetectable UndetectableUndetectable (OH-Lysine-1) Hydroxy-Lysine-2 1.0 UndetectableUndetectable Undetectable Undetectable (OH-Lysine-2) Homocystine 1.0Undetectable Undetectable ≤2.5 ≤2.1 Phenylalanine UNK 4-31 4-14 ≤2.56-31 Tryptophan 1.0 ≤5.9 ≤7.7 0.6-4.6  ≤9.3 Ornithine 2.0 ≤25.7  ≤4.5≤4.7 ≤14.2  Lysine UNK 6-38 3-29 9-58 19-60 

Example 5—Quantification of Amino Acid Content in Saliva

Amino acid analysis was performed on saliva samples of nine (9) adults(3 males and 6 females). All individuals were assessed as clinicallynormal and were fully ambulatory, community dwelling, healthy, and on nomedications. Table 4 provides the actual measured ranges of amino acidlevels. For diagnostic purposes, these ranges may be considered “normal”ranges). The normal range for amino acids which are undetectable shouldbe taken to be less than or equal to the LOQ.

TABLE 4 Adult Amino Acid Levels in Saliva Amino Acid Normal RangeAspartic Acid 2.6-9.2  Arginosuccinic Acid (ASA) UndetectableSulfo-Cysteine ≤1.3 Glutamic Acid 1.5-26.0 Hydroxyproline (OH-Proline)≤1.6 Serine 0.9-4.9  Asparagine Undetectable Phospho-Ethanolamine (PEA)≤133.8 α-Amino Adipic Acid (AAD) 0.4-2.8  Glycine 24.5-425.9 Glutamine0.7-20.2 Sarcosine 0.4-11.0 Histidine 4.3-59.8 β-Alanine (BALA) ≤6.0Taurine 26.5-177.5 Citrulline 1.5-21.7 Carnosine 0.0 Threonine 0.1-0.4 Arginine 4.9-26.4 Anserine ≤0.1 1-Methylhistidine (1-Me-His) ≤0.83-Methylhistidine (3-Me-His) ≤0.4 Alanine 7.6-39.6 γ-Amino-Butyric Acid(GABA) 0.4-2.4  β-Amino-Isobutyric Acid (BAIB) ≤1.0 Proline 10.5-264.1Ethanolamine 5.1-42.3 α-Amino-Butyric Acid (AAB) ≤0.6 Tyrosine 8.5-63.1Valine 0.3-7.9  Methionine 0.4-0.9  Cystathionine-A (Cysta-A)Undetectable Cystathionine-B (Cysta-B) Undetectable Cystine ≤3.7Isoleucine 0.4-1.7  Allo-Isoleucine (allo-Ile) Undetectable Leucine0.2-6.1  Hydroxy-Lysine-1 (OH-Lysine-1) ≤0.1 Hydroxy-Lysine-2(OH-Lysine-2) Undetectable Homocystine Undetectable Phenylalanine3.8-31.5 Tryptophan ≤1.2 Ornithine 4.8-72.0 Lysine 6.2-81.0

All patents and other references cited in the specification areindicative of the level of skill of those skilled in the art to whichthe invention pertains, and are incorporated by reference in theirentireties, including any tables and figures, to the same extent as ifeach reference had been incorporated by reference in its entiretyindividually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to obtain the ends and advantages mentioned,as well as those inherent therein. The methods, variances, andcompositions described herein as presently representative of preferredembodiments are exemplary and are not intended as limitations on thescope of the invention. Changes therein and other uses which will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

Also, unless indicated to the contrary, where various numerical valuesare provided for embodiments, additional embodiments are described bytaking any two different values as the endpoints of a range. Such rangesare also within the scope of the described invention.

Thus, additional embodiments are within the scope of the invention andwithin the following claims.

What is claimed is:
 1. A method for determining the amount of at leasttwo amino acids in a sample by tandem mass spectrometry, the methodcomprising: (a) purifying the at least two amino acids comprisingtaurine and beta alanine by liquid chromatography (LC); and (b) ionizingthe at least two amino acids under conditions suitable to produce ionsdetectable by mass spectrometry; (c) detecting the amount of ions of theat least two amino acids by tandem mass spectrometry (MS/MS); (d)determining the amount of the at least two amino acids in the samplefrom the amount of ions determined in step (c).
 2. The method of claim1, wherein the at least two amino acids further comprise alloisoleucine.3. The method of claim 1, wherein the at least two amino acids furthercomprise aspartic acid, threonine, serine, asparagine, glutamic acid,glutamine, glycine, alanine, citrulline, valine, cystine, methionine,isoleucine, leucine, tyrosine, phenylalanine, gamma-amino-butyric acid,lysine, and histidine.
 4. The method of claim 1, wherein the at leasttwo amino acids further comprise aspartic acid, hydroxyproline,threonine, serine, asparagine, glutamic acid, glutamine, sarcosine,alpha-amino-adipic acid, proline, glycine, alanine, alpha-amino-butyricacid, valine, cysteine, methionine, homocitrulline, cystathionine,alloisoleucine, isoleucine, leucine, tyrosine, phenylalanine,arginosuccinic acid, beta-amino-isobutyric acid, homocystine,gamma-amino-butyric acid, tryptophan, hydroxylysine, lysine, andhistidine.
 5. The method of claim 1, wherein the sample is urine.
 6. Themethod of claim 1, wherein the sample is plasma.
 7. The method of claim1, wherein the amount of the two or more amino acids is used to diagnoseor monitor one or more diseases selected from the group consisting ofMaple Syrup Urine Disease, kidney failure, Crohn's disease, ulcerativecolitis, chronic fatigue syndrome, Wilson's disease, Cushing's disease,gout, and hyperactivity disorders.
 8. The method of claim 1, wherein themethod comprises using one or more internal standards.